A new MYH2 variant in an Italian patient expanding the clinical spectrum of MYH2-related myopathy

Abstract

Background

Myosin heavy chain (MyHC)-related congenital myopathies display variable age of onset and clinical manifestations depending on the mutated isoform. Cardiomyopathy, ophthalmoplegia and primarily proximal weakness may be part of the clinical picture.

Case presentation

A 57-year-old male patient with a history of arterial hypertension and hyperferritinemia (thalassemic trait) began to experience lower limb proximal weakness at the age of 23 years and he got progressively worse over the years when he also reported mild dyspnea, easy fatigability, fasciculations and a sort of discomfort in the posterior muscles of both thighs even at rest. He had no diplopia or dysphagia.

Peculiar clinical features were bilateral exophthalmos, slight eyelid ptosis, limited ocular motility in all gaze directions, marked lower limb proximal weakness with posteromedial thigh hypotrophy and hypertrophic calves with increased consistency. Severe signs of both myopathic and neurogenic degeneration were seen at quadriceps skeletal muscle biopsy.

Serum CK values were slightly elevated (less than 300 U/L). His parents are second-degree cousins and have normal serum CK levels, a proband’s first cousin had a diagnosis of muscular dystrophy and died at the age of 60 years, wheelchair bound. A 46-year-old sister is healthy.

EMG showed signs of reinnervation in all muscles, myopathic signs being evident in the gastrocnemius muscles. Clear signs of fibro-adipose degeneration were observed at muscle MRI, more evidently so in the semitendinosus, rectus femoris, sartorius, gracilis and gastrocnemius muscles.

Conclusions

Genetic analysis revealed the new homozygous variant c.3901G > T in exon 29 of MYH2 gene (NM_017534), predicted to introduce the premature stop codon p.(Glu1301*), thus leading to a diagnosis of MYH2-related myopathy. This variant shows a clear genotype-phenotype correlation, as it leads to a near-complete loss of MyHC IIa expression and results in a recessive myopathy characterized by adult-onset progressive muscle weakness with ophthalmoplegia and myopathic changes consistent with biallelic truncating mutations.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12883-026-04790-z.

Introduction

Six skeletal muscle myosin heavy chains (èMYH1, MYH2, MYH3, MYH4, MYH8, and MYH13) are encoded by genes found in a tightly linked cluster on chromosome 17 [1, 2], whereas MYH6 and MYH7, which encode cardiac myosin isoforms, are located on chromosome 14. Among the diverse isoforms of myosin expressed in skeletal muscle, MYH2 is pivotal in determining muscle fiber type and function.

The MYH2 expression is tightly regulated during muscle development and adaptation, its abundance and functional diversity being influenced by various physiological and environmental factors – including exercise, aging, and disease – as well as by its structural features and by post-translational modifications and interactions with other proteins within the sarcomere. MYH2 encodes a myosin heavy chain isoform essential for the contractile properties of fast-twitch oxidative (type IIa) muscle fibers [3].

Congenital myopathies linked to variants in skeletal muscle myosin heavy chain genes represent a rare category of hereditary muscular disorders with variable age of onset and clinical manifestations depending on the physiological role and expression pattern of the affected gene and on the severity of variants [4].

Numerous pathological variants, responsible for skeletal myopathies associated with cardiomyopathy, have been found in MYH7 gene [5]. MYH2-associated variants are much less frequent [6], occurring in both autosomal dominant and recessive forms and resulting in congenital myopathies clinically characterized by ophthalmoparesis and primarily proximal weakness.

The main myopathological feature of MYH2-related myopathies is the loss and/or atrophy of type 2 A fibers, additional findings possibly including dystrophic changes, minicores, intranuclear or cytoplasmic inclusions. Protein aggregation, in the form of tubulofilamentous inclusions in association with vacuolated muscle fibers, is a hallmark of myosinopathies [4]. Clinically, both proximal and, sometimes, distal weakness and joint contractures are reported, without any clear correlation with the mode of inheritance.

We now report the case of 57-year-old male patient carrying the new homozygous c.3901G > T variant in MYH2 gene (NM_017534) likely introducing a premature stop codon p.(Glu1301*) in the myosin tail domain, expanding the spectrum of recessive forms of MYH2-related myopathy, formerly Congenital Myopathy-6 with ophthalmoplegia (MIM # 605637).

Case presentation

Clinical aspects

The proband is a 57-year-old male patient with a history of pharmacologically well-managed arterial hypertension, hyperferritinemia associated with thalassemic traits and heterozygosity for the p.His63Asp variant of the HFE (homeostatic iron regulator; MIM*613609) gene causing hemochromatosis (MIM # 235200). His parents are second-degree cousins, and, also, a patient’s first-degree cousin was affected with an unspecified form of muscular dystrophy and had died at the age of 60 years after years of wheelchair dependence. The patient, who has no offspring, has a healthy 46-year-old sister.

At the age of 23, the patient began to experience lower limb weakness, progressively worsening over the years. He now reports significant difficulty in climbing and descending stairs, inability to rise independently from a sitting or squatting position. He refers mild dyspnea, rapid exhaustion, fasciculations, but no diplopia or dysphagia. Additionally, he persistently feels “discomfort” on the posterior side of both thighs, even at rest.

Neurologic examination revealed bilateral exophthalmos, mild bilateral eyelid ptosis, and limited ocular motility in all gaze directions, more pronounced upwards. Upper limb strength was preserved, except for mild weakness in both triceps brachii and interosseous on the right side. No signs of muscle hypotrophy or winged scapula were observed. Manual muscle testing following Medical Research Council (MRC) scale, showed bilateral weakness in iliopsoas (MRC 4) and leg abductors bilaterally, marked hypotrophy in both thighs (particularly in the medial area), whereas both calves were hypertrophic and of markedly increased (“stony”) consistency. Osteotendinous reflexes were present and symmetrical in all four limbs. Walking on tiptoes and heels was good.

His serum creatine kinase (CK) values were never higher than 274 U/L (normal values were found in the father, 126 U/L, and the mother, 146 U/L).

The patient underwent the first electromyography (EMG) at the age of 57 years; there were chronic radicular signs with normal nerve conduction velocities and no myopathic signs. A few months later, signs of reinnervation were seen in all examined muscles with the exception of gastrocnemius muscle in which there was a coexistence of myopathic signs and silent areas, the latter indicating area lacking electrical activity during contraction. Muscle Magnetic Resonance Imaging (MRI) displayed extensive fibro-adipose degeneration without any evidence of edema suggesting a primary non-inflammatory myopathy. Specifically, the degeneration was prominent in the pelvic girdle – mostly in the gluteus maximus - all paravertebral muscle and in the triceps brachii without associated atrophy. Also, in the thigh and leg muscles there were clear signs of muscle tissue replacement, more evidently so in the semitendinosus, rectus femoris, sartorius, gracilis and gastrocnemius muscles (Fig. 1 Supplementary).

Based on MRI results, the patient underwent right vastus lateralis skeletal muscle biopsy in our Neuromuscular Center.

Results

Muscle biopsy findings

Light microscopy examination of skeletal muscle tissue (right vastus lateralis) showed important fiber size variability due to the presence of markedly hypotrophic and/or hypertrophic fibers. Some fibers displayed hyperchromic, intracytoplasmic areas distinctly separated from the surrounding cytoplasm, suggesting potential initial degeneration. We observed several, single or multiple, nuclear centralizations as well as nuclear clumps and fiber splittings. In addition, numerous fibers presented one or more intracytoplasmic vacuoles often with fuchsinophile reinforcement and containing either hyperchromic or slightly dyeable material. No necrotic fibers or interstitial cellular infiltrates were identified. Endomysial connective tissue was slightly increased, initial fatty replacement being also noted (Fig. 1A, B). Several fibers showed centrally rarefied NADH-tetrazolium reductase activity indicating a disorganization of the intermyofibrillar network (Fig. 1C). We also detected rare COX-deficient fibers (Fig. 1D). Both glycogen and lipid content were normally represented (data not shown). ATPase staining revealed a prevalence of type I fibers (Fig. 1E) compared to control (Fig. 1F), a distribution confirmed by immunofluorescence staining with slow-MHC antibody (Fig. 1G) compared to control (Fig. 1H). Lillie’s stain ruled out the presence of iron accumulation in skeletal muscle (data not shown).

Fig. 1.

Muscle biopsy. H&E (A) and MTG (B) show fiber size variability, the presence of hyperchromic intracytoplasmic areas (asterisk at the inset), vacuoles with fuchsinophilic subsarcolemmal reinforcement. NADH (C) and COX (D) activities are centrally rarefied in some fibers. ATPase at pH 4.6 (E) shows the prevalence of type I fibers compared to normal fiber type distribution in control muscle (F). Immunofluorescent staining for slow-MHC confirms the prevalence of type I fibers (G) compared to control (H). Membrane fibers were counterstained with caveolin-3 and nuclei with DAPI. Scale bar 50 μm

Gene and protein study

Clinical exome sequencing revealed the presence of the novel homozygous MYH2 nucleotide change chr17-10430344 C-A, corresponding to the c.3901G > T variant (NM_017534.6), likely resulting in the premature stop codon p.(Glu1301*) (Fig. 2A). The variant was absent in population databases (GnomAD MAF:0) and classified as Likely Pathogenic, according ACMG criteria. The variant was found heterozygous in the unaffected patient’s parents, supporting its biallelic inheritance.

Fig. 2.

Genetic and protein findings. A Electropherogram showing the homozygous nucleotide change c.3901G > T in our patient and segregation analysis in his unaffected parents. B Analysis of the relative level of expression of different isoforms of MyHC mRNA in patient’s and control’s muscles (mean of two independent PCR analysis). C Immunofluorescent staining for MyHC-IIa in patient and control; laminin-α2 for membrane staining and DAPI for nuclei counterstaining. Scale bar 50 μm. D Western blot analysis of MyHC-IIa levels in patient’s and control’s muscles

Relative expression of myosin heavy chain isoforms revealed very low levels of MyHCIIa mRNA and a predominant expression of MyHCI mRNA in the patient compared to the control (mean of two independent PCR analyses) (Fig. 2B). Quantification of MyHC-IIa protein expression by MyHC-IIa immunofluorescence staining showed very few positive fibers in our patient compared to control (Fig. 2C). Western blot analysis showed complete absence of the MyHC-IIa protein related band in our patient (Fig. 2D).

Immunofluorescence staining of protein aggregates

The staining for autophagic marker p62 showed an increased signal in some fibers, either concentrated in restricted areas or widely scattered in the cytoplasm of the fiber (Fig. 3A, B). αB-crystallin staining detected cytoplasmatic protein deposits/aggregates, sometimes of considerable size, in some fibers (Fig. 3C, D). Positivity was also observed in the interstitial area of muscle sample indicating a diffuse activation of autophagic activity in our patient. TDP-43 staining displayed spot protein deposition in some fibers around centralized nuclei and in cytoplasmic areas delimited by caveolin-3-positive membrane (Fig. 3E, F). Some intracytoplasmic vacuoles positively stained with membrane caveolin-3 antibody which also revealed the presence of scattered cytoplasmic aggregates (Fig. 3G) and of small sarcolemmal invaginations inside the cytoplasm (Fig. 3H), the latter being absent when using anti-dystrophin antibodies (data not shown).

Fig. 3.

Immunofluorescence of protein aggregates. p62 positivity in some fibers (A). B: Control muscle staining for p62. αB-crystallin-positive protein aggregates of variable size in some fibers (C). D: Control muscle staining for αB-crystallin. TDP-43 evidence of cytoplasmic aggregates delimited by caveolin-3-positive membrane (E). F: Control muscle staining for TDP-43. Membrane alterations and scattered cytoplasmic aggregates detected with caveolin-3 membrane staining (G, H). Scale bar 50 μm. Immunofluorescence of sarcomeric proteins. Immunofluorescence staining for α-actinin (I), nebulin (K) and titin (M, O) in patient and control (J, L, N, P) muscle. Caveolin-3 (red) and DAPI (blue) counterstained membranes and nuclei. Scale bar 50 μm

Immunofluorescence staining of sarcomeric proteins

The staining for α-actinin evidenced the presence of positivity and a residual partial sarcomeric organization which takes on a “disheveled” appearance (Fig. 3I, J). Nebulin staining confirmed the almost complete disappearance of sarcomeric organization compared to normal muscle (Fig. 3K, L). Titin staining was highly inhomogeneous, an indication of a markedly altered cytoskeletal architecture (Fig. 3M, N) which was confirmed by testing the antibody on longitudinal sections (Fig. 3O, P).

Electron microscopy

Ultrastructural examination revealed significant alterations in the sarcomeric structure of several muscle fibers, as illustrated in Fig. 4. Specifically, we observed Z-band streaming (Fig. 4A), along with the presence of aggregates with an electron-dense granulofilamentous appearance (Fig. 4B). Additionally, filamentous bodies were detected (Fig. 4C). In several other fibers, we discerned areas characterized by a complete disarray of filaments and fragmentation of the Z line. This disarray was sometimes accompanied by the initial formation of nemaline bodies (Fig. 4D). Intranuclear filaments were not observed.

Fig. 4.

Ultrastructural findings. A Alterations in the sarcomeric structure observed at ultrastructural examination: Z-band streaming (A), accumulation of electron-dense granulofilamentous aggregates (B), presence of filamentous bodies (C). Areas of filament disarray and Z line fragmentation with initial nemaline body formation (D). Scale bar: 715 nm (B-C)- 1135 nm (A-D)

Discussion

Hereditary myosin myopathies have emerged as an important group of diseases with variable clinical and morphological expression dependent on the mutated isoform and the type and location of the genetic variants [7]. Myosin myopathy with external ophthalmoplegia is associated with variants in the fast myosin heavy chain gene MYH2 that is expressed in type 2 A muscle fibers and is inherited in either a dominant or a recessive manner [4].

The first reported patients with recessive myosin IIa myopathy [8] carried compound heterozygous or homozygous truncating variants in MYH2. Several additional cases carrying either truncating or missense variants have later been reported [9]. In addition to muscle weakness, which is mostly proximal, all the described individuals presented external ophthalmoplegia occasionally associated with ptosis and facial muscle weakness (Fig. 5, Supplementary Tables 1 and Fig. 2 Supplementary).

Fig. 5.

Schematic representation of MYH2 gene structure, protein domains and distributions of reported pathogenic MYH2 variants with recessive (AR, above the scheme of the gene) or dominant (AD, under the scheme of the gene) inheritance

Less than 70 patients have been described harboring pathogenic variants in MYH2 and half of them belong to two large pedigrees: the Swedish “dominant” family (n = 19 patients) [10, 11] and large “recessive” Arab kindred (n = 16 patients) [12–14]. Considering available clinical data (displayed in Additional Table 1), most of the patients (> 90%) displayed a pediatric onset, one third of them with a congenital/neonatal presentation. Adult-onset cases are rare (< 10%).

Despite heterogeneity in the case reports available (please refer to Additional Table 1), up to 90% of individuals affected by MYH2-related myopathy displayed PEO accompanied with ptosis in 46% of subjects. Ptosis without PEO was extremely rare. Extraocular muscle weakness was also frequent with additional involvement of pelvic girdle (and/or lower limbs) in 81% of cases and of shoulder girdle (and/or upper limbs) in 78% of patients. Facial and neck weakness were also frequently reported (64% and 45%, respectively). Interestingly, hand weakness and tremor were reported in about 20% of mutated subjects. Cardiorespiratory involvement is rare. The presence of abnormal spinal curves and contractures is restricted to the members of the first autosomal dominant familial case [10]. Elevated serum CK levels and myogenic findings at EMG were found in more than half of the patients in which these investigations were performed (59% and 70%, respectively).

So far, 23 molecular defects in MYH2 have been described (Supplementary Table 2): nine missense changes, eight variants predicted to alter reading frame (three indels and five splice-site variants) and six SNVs introducing a premature stop codon. Pathogenic variants acting in a dominant fashion were four, two of them reported as de novo variants [15–18] and the other two displaying full penetrance across family members [11, 19, 20]. Three of the four dominant variants are in the tail domain (intron 39 or exon 39) while recessively inherited variants are scattered along MYH2 gene, although a higher number of variants is detected in the motor head domain (n = 12) followed by coiled coil tail and neck domains (n = 4 and n = 3, respectively).

Available clinical data can be stratified according to patient’s genotype considering dominant cases and recessive forms. In recessive forms, patients with biallelic nonsense variants (“null” genotype) can be distinguished from patients presenting mono or bi-allelic missense variants.

Although the percentage of patients with pediatric onset is similar, congenital and neonatal onset largely predominate in the dominant cases. Considering clinical symptoms, girdle and limb weakness is slightly increased in the dominant forms while ptosis, facial muscles and neck flexor weakness are more frequent in recessive cases. In the latter, the frequency of ptosis in patients with a null genotype is approximately twice that observed in those carrying at least one missense allele. As said, contractures at birth have been only observed in dominant cases with congenital onset. Hand weakness (42%) and tremor (51%) were almost exclusively reported in dominant cases. Scoliosis was reported in half of the recessive cases and is unlikely in the dominant forms.

In this study, we report the case of a 57-year-old male patient carrying the homozygous c.3901G > T variant in exon 29 of MYH2 gene causing a premature stop codon p.(Glu1301*), resulting in a drastic loss of expression of MyHC IIa mRNA as well as of MYH2 protein. Considering the variant type, its absence from control databases, its recessive inheritance (supported by consanguinity between patient’s parents) and evidence of its impact on MYH2 transcript and protein levels, the c.3901G > T variant can be re-classified as Pathogenic (PVS1, PS3, PM2).

Our patient presented an adult onset, progressively worsening lower limb weakness without contractures, significant difficulty climbing and descending stairs, inability to rise independently from a seated to an upright position. In addition, he had bilateral exophthalmos, mild bilateral eyelid ptosis, and limited ocular motility in all gaze directions, matching the main clinical aspects observed in recessive patients with biallelic truncating variants.

He carries the H63D variant in the HFE gene, which is associated with hereditary hemochromatosis, a disorder characterized by excessive iron absorption by the intestines. The specific effects of heterozygosity for this variant on skeletal muscle are not as well-studied as its effects on other tissues. Typically, hereditary hemochromatosis primarily affects organs such as liver, heart, pancreas, and endocrine glands where iron accumulates, leading to damage and dysfunction. However, skeletal muscle can also be affected by iron overload in cases of severe hemochromatosis because the HFE protein is expressed at low levels in skeletal muscle tissue (Human Protein Atlas). In individuals heterozygous for the H63D variant, the effects on skeletal muscle are expected to be less pronounced compared to those who are homozygous for the variant or have compound heterozygosity with other hemochromatosis-associated variants. Nonetheless, even a low iron overload can potentially lead to oxidative stress and mitochondrial dysfunction in skeletal muscle cells, contributing to muscle weakness, fatigue, sarcopenia, frailty, and chronic pain [21, 22]. For this reason, we used Lillie’s stain to evaluate iron content in skeletal muscle tissue, but we did not observe any significant iron deposition.

Patient’s muscle biopsy showed an almost complete absence of type II fibers and myopathic changes including fiber size variability, centralized nuclei, increased connective and adipose tissue, rimmed vacuoles and protein aggregates. Also, the oxidative enzymatic activity was more faintly distributed in the center of several fibers giving them a core-like aspect.

Some clinical features – namely the late and progressive onset – as well as the morphological evidence of dystrophic changes and the presence of rimmed vacuoles remind of a dominantly inherited form, whereas the lack of contractures, the marked reduction of MyHC-IIa and 2 A fibers and the presence of core-like fibers are typical of a recessive form.

A patient with a homozygous recessive variant, but with more autosomal-dominant clinical and histological aspects has already been reported by Findlay in 2018 in a family carrying the homozygous recessive MYH2 variant c.737G > A causing the p.Arg246His change [16, 23].

In 2020, Telese and colleagues [9] described a patient with adulthood disease onset, progressive muscle weakness, elevated CK levels, ophthalmoplegia, mild facial weakness, and histological features recalling those described by Findlay and colleagues [16, 23]. However, the absence of congenital contractures and the pattern of diffuse weakness were more similar to those described for AR patients. This patient carried two heterozygous truncating variants: p.Arg793Ter, which is located in the myosin motor domain and interrupts the protein 1148 amino acids before the natural stop codon, and p.Glu1461Ter, which interrupts the coiled coil rod domain 480 amino acids before the stop codon. To date, no other variants have been reported on exon 29. At the age of 60 years the patient showed ptosis, ophthalmoplegia and mild proximal muscle weakness. MRI showed prominent fatty infiltration in quadriceps, adductors and semitendinosus muscles. His quadriceps muscle biopsy showed fatty infiltration, type 1 fiber uniformity and numerous fibers with internalized nuclei.

Interestingly, even in the presence of premature stop codons, both our patient and the one reported by Telese et al. [9] still have residual protein expression as demonstrated by the immunofluorescence: we hypothesize it could be a non-functional truncated protein anyway recognized by the antibody. Indeed, no signal for MHY2 protein was detected by Western blot analysis. Our results in immunofluorescent staining showed highly inhomogeneous signals for both titin and nebulin. Titin interacts with the myosin thick filaments and maintains their alignment within the sarcomere, nebulin acts as a molecular ruler that regulates the length of actin filaments, thereby ensuring proper alignment and interaction with MyHC-IIa during muscle contraction. This interaction helps ensure optimal overlap between actin and myosin filaments for effective contraction. Similarly to titin, nebulin staining confirmed the almost complete disappearance of sarcomeric organization compared to normal muscle. We speculated that this new variant in the myosin tail domain of MYH2 affects myofibril organization and the sarcomeric structure.

Our observations suggest that, though some features remain well-defined, there is no clear-cut clinical and histological distinction between recessive and dominant forms and, similarly, between the phenotype and the domain, namely the motor domain or the myosin one, in which the variant falls.

MYH2-related myopathy exhibits substantial phenotypic overlap between recessive and dominant forms, with no strict correlation to variant type or protein domain. This adult-onset case with a homozygous truncating MYH2 variant broadens the spectrum of recessive disease and underscores the clinical and histological variability of MYH2 myopathies.

Abbreviations

CK

Creatine kinase

MyHC

Myosin heavy chain

ATP

Adenosine triphosphate

H&E

Hematoxylin and eosin

MGT

Modified Gomori trichrome

COX

Cytochrome c oxidase

SDH

Succinate dehydrogenase

NADH

Nicotinamide adenine dinucleotide reductase

PAS

Periodic acid Schiff

NGS

Next generation sequencing

HFE

Homeostatic iron regulator

EMG

Electromyography

MRI

Magnetic resonance imaging

MRC

Medical Research Council

PEO

Progressive external ophthalmoplegia

PCR

Polymerase chain reaction

RT-PCR

Reverse transcription PCR

Authors’ contributions

M.S., F.M. and D.V performed clinical analysis and collected clinical data; S.Z., M.S., D.R edited the manuscript; D.R., S.P. and R.D.B performed genetic analysis; S.Z., M.P. and L.N performed morphological studies; P.C: and L.B. contributed technical support; S.C. and G.P.C revised the manuscript for intellectual content. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Italian Ministry of Health, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico Ricerca Corrente 2024 to GPC.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from patient to publish this Case Report.

Competing interests

The authors declare no competing interests.